Attenuated live Neospora vaccine

ABSTRACT

The present invention provides attenuated live cultures of the pathogenic protozoan parasite,  Neospora,  and live vaccines against neosporosis prepared therefrom which are useful in the prevention of clinical disease and abortion in mammals.

FIELD OF THE INVENTION

The present invention relates to attenuated strains of the pathogenicprotozoan, Neospora, and to live vaccines against neosporosis preparedfrom the attenuated strains which are useful in the prevention ofclinical disease and abortion in mammals.

BACKGROUND OF THE INVENTION

Neospora is a pathogenic protozoan parasite of animals which hasrecently been recognized as a major cause of abortion, neonatal death,congenital infection, and encephalitic disease in mammals. Dubey andLindsay, 1993, Parasitology Today, 9:452-458. N. caninum infects dogs,and congenitally infects pups, often leading to paralysis. Tachyzoitesof N. caninum have been isolated from naturally infected pups. Lindsayand Dubey, 1989, J. Parasiol. 75:163-165. Neospora spp. are a majorcause of abortion in dairy cattle. Cases of Neospora-related disease,i.e., neosporosis, have also been reported in goats, sheep and horses.

Although N. caninum is superficially similar to the pathogen, Toxoplasmagondii, N. caninum and T. gondii have been distinguished from each otherantigenically and ultrastructurally. Dubey and Lindsay, 1993, above. Inaddition, Neospora-like protozoal parasites isolated from the brains ofaborted bovine fetuses and continuously cultured in vitro were shown tobe antigenically and ultrastructurally distinct from both T. gondii andHammondia hammondi, and most similar to N. caninum. Conrad et al., 1993,Parasitology 106:239-249. Furthermore, analysis of nuclear small subunitribosomal RNA genes revealed no nucleotide differences between Neosporaspp. isolated from cattle and dogs, but showed consistent differencesfrom T. gondii. Marsh et al., 1995, J. Parasitol. 81:530-535.

The etiologic role of a bovine isolate of Neospora in bovine abortionand congenital disease has been confirmed. Barr et al., 1994, J. Vet.Diag. Invest. 6:207-215. A rodent model of central nervous systemneosporosis has been developed using inbred BALB/c mice infected with N.caninum. Lindsay et al., 1995, J. Parasitol. 81:313-315. In addition,models to study transplacental transmission of N. caninum in pregnantoutbred and inbred mice have been described by Cole et al., 1995, J.Parasitol. 81:730-732, and by Long et al., 1996, J. Parasitol.82:608-411, respectively. Furthermore, an experimental N. caninum pygmygoat model that closely resembles naturally acquired Neospora-inducedcattle abortion has been demonstrated. Lindsay et al., 1995, Am. J. Vet.Res. 56:1176-1180.

WO 9525541 discloses a biologically pure culture of bovine Neospora ,methods of detecting anti-Neospora antibodies and Neospora-specificnucleic acids, and a composition containing a bovine Neospora antigenand carrier for use as a vaccine. WO 9525541 does not, however, teachattenuated live cultures of Neospora , or live vaccines preparedtherefrom which are able to trigger a protective immune response in avaccinated animal.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides cultures of cells of astrain derived from a pathogenic parent strain of a species of Neospora, which cells exhibit attenuated pathogenicity compared to those of theparent strain but which are capable of triggering an immune responsethat protects a mammal against neosporosis when administered as a livevaccine.

In a second aspect, the present invention provides vaccines to protect amammal against neosporosis, comprising an immunologically effectiveamount of live cells of a strain derived from a pathogenic parent strainof a species of Neospora, which cells exhibit attenuated pathogenicitycompared to those of the parent strain but which are capable oftriggering an immune response that protects the mammal againstneosporosis when administered as a live vaccine, and a veterinarilyacceptable carrier. Vaccines of the invention may further comprise oneor more other components including, for example, an adjuvant. Vaccinesof the present invention may be administered to any mammalian speciessusceptible to infection and disease caused by Neospora including, butnot limited to, dogs, cows, goats, sheep and horses.

In a third aspect, the present invention provides methods for preparingcultures of attenuated cells from a pathogenic strain of Neospora foruse in a vaccine that protects a mammal against neosporosis, comprisingmodifying cells from a pathogenic parent strain of a species ofNeospora; selecting and clonally propagating one or more modified cellsthat exhibit attenuated pathogenicity compared to cells of the parentstrain; and selecting and clonally propagating one or more attenuatedcells which are capable of triggering an immune response that protectsthe mammal against neosporosis when administered in a live vaccine.

In a fourth aspect, the present invention provides methods for preparinga vaccine that protects a mammal against neosporosis, comprisingmodifying cells from a pathogenic parent strain of a species ofNeospora; selecting and clonally propagating those modified cells thatexhibit attenuated pathogenicity compared to cells of the parent strainbut which are capable of triggering an immune response in the mammalthat protects against neosporosis when administered in a live vaccine;and combining an immunologically effective amount of the attenuatedcells with a veterinarily acceptable carrier in a form suitable foradministration as a live vaccine to the mammal.

In a fifth aspect, the present invention provides methods forvaccinating a mammal against neosporosis, comprising administering tothe mammal an immunologically effective amount of a vaccine comprisinglive cells of a strain derived from a pathogenic parent strain of aspecies of Neospora, which cells exhibit attenuated pathogenicitycompared to those of the parent strain but which are capable oftriggering an immune response that protects the mammal againstneosporosis when administered as a live vaccine, and a veterinarilyacceptable carrier.

In a sixth aspect, the present invention provides combination vaccines,comprising an immunologically effective amount of live cells of a strainderived from a pathogenic parent strain of a species of Neospora, whichcells exhibit attenuated pathogenicity compared to those of the parentstrain but which are capable of triggering an immune response thatprotects the mammal against neosporosis when administered as a livevaccine; one or more other antigens that trigger an immune response thatprotects the mammal against a disease or a pathological condition; and aveterinarily acceptable carrier. The combination vaccines may furthercomprise one or more other components including, for example, anadjuvant.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have discovered that cells of a pathogenic strain of aspecies of Neospora may be attenuated, and that the resulting attenuatedcells are capable of triggering an immune response that protects mammalsagainst neosporosis when administered as a live vaccine. The presentinvention thus provides cultures of cells of a strain derived from apathogenic parent strain of a species of Neospora, which cells exhibitattenuated pathogenicity compared to those of the parent strain butwhich are capable of triggering an immune response that protects amammal against neosporosis when administered as a live vaccine.

The present invention further provides methods for preparing cultures ofattenuated cells of a species of Neospora for use in a vaccine thatprotects a mammal against neosporosis, comprising modifying cells from apathogenic parent strain of a species of Neospora, for example, by highserial passage, or by exposure to a mutagenic agent, or by geneticengineering using recombinant DNA techniques; selecting and clonallypropagating one or more modified cells that exhibit attenuatedpathogenicity compared to cells of the parent strain; and selecting andclonally propagating one or more attenuated cells which are capable oftriggering an immune response that protects the mammal againstneosporosis when administered in a live vaccine.

As used herein, the term “neosporosis” refers to infection of a mammalby a species or strain of Neospora, or to any clinical symptom,condition, event or pathology associated with infection of the mammal byNeospora.

The term “attenuated” as used herein describes a cell, culture, orstrain of Neospora exhibiting a detectable reduction in infectivity orvirulence in vitro or in vivo as compared to that of the parent strainof Neospora from which the attenuated cell, culture, or strain isderived. Reduction in virulence encompasses any detectable decrease inany attribute of virulence, including infectivity in vitro or in vivo,or any decrease in the severity or rate of progression of any clinicalsymptom or condition associated with infection.

The term “parent strain” refers to a strain of Neospora which exhibits arelatively higher degree of pathogenicity when administered to a mammalthan an attenuated strain which is derived therefrom by one or morepassages in vivo or in vitro and/or one or more attenuation steps.

The present invention further encompasses preparation and use in avaccine of cells of a strain of Neospora derived from a strain orspecies that is not pathogenic in a particular mammalian species, butwhich cells have been modified by chemical or genetic means to becapable of triggering a protective immune response in members of thatmammalian species.

The live attenuated cells of the invention are capable of triggering animmune response that protects a mammal against neosporosis after one ormore administrations as a live vaccine. A “protective immune response”is defined as any immunological response, either antibody or cellmediated immunity, or both, occurring in the mammal that either preventsor detectably reduces subsequent infection, or eliminates or detectablyreduces the severity, or detectably slows the rate of progression, ofone or more clinical symptoms or conditions associated with neosporosis.The term “immunologically effective amount” refers to that amount ordose of vaccine or antigen that triggers a protective immune responsewhen administered to a mammal.

Preparation of Attenuated Strains of Neospora

Since the invention is based on the discovery that cells of a pathogenicstrain of Neospora may be attenuated, and that the resulting attenuatedcells are capable of triggering an immune response that protects amammal against neosporosis when administered as a live vaccine, practiceof the invention is not limited to any particular method of attenuation.Rather, attenuation of cells of a pathogenic strain of Neospora may becarried out by any techniques or procedures known in the art including,but not limited to, high serial passage, or exposure to a mutagenicagent, or by genetic engineering using recombinant DNA technology, orsome combination thereof.

High serial passage may be carried out by repeated in vitro passaging ofcells of a pathogenic strain of Neospora in susceptible host cells untilsufficient attenuation occurs. Passaging may be conducted under specificenvironmental conditions to select for attenuated cells. For example,passaging may be conducted at a temperature below that of the bodytemperature of the intended mammalian vaccinate to select fortemperature-sensitive strains of Neospora that will not grow, or thatwill only grow at a reduced rate, when administered in a vaccine to themammal.

Mutagenesis may be carried out by exposure of Neospora cells to either achemical mutagen or to radiation, as described in the art. Anon-limiting example of a chemical mutagen useful in the practice of theinvention is N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (Sigma), theuse of which is described below in Example 1. Radiation may be selectedfrom either ultraviolet light or ionizing radiation.

The degree of exposure to the mutagen, i.e., the concentration ofchemical mutagen, or the level of radiation, as well as the duration ofexposure, is preferably that amount which results in producing one ormore viable cells of Neospora that exhibit an attenuated level ofpathogenicity but that are capable of triggering an immune response thatprotects against neosporosis when administered as a live vaccine to amammal. Appropriate parameters for use of mutagenic agents may bedetermined empirically using standard techniques.

Pathogenic strains of Neospora may also be attenuated using recombinantDNA technology according to techniques known in the art, and the presentinvention is intended to encompass such modified strains and vaccinesprepared therefrom. Non-limiting examples of recombinant DNA techniqueswhich may be used to practice the invention include gene replacement orgene knockout to disable one or more genes, resulting in a strain havingan attenuated pathogenicity. Genes that may be disabled include, forexample, an essential metabolic gene, or a gene encoding a virulencefactor, or a gene encoding a surface antigen that plays a role inmodulating the immune response in the mammalian host

A non-limiting example of an essential metabolic gene that may usefullybe targeted for disruption in the Neospora genome is the dihydrofolatereductase-thymidylate synthase (DHFR-TS) gene. Titus et al., 1995, Proc.Natl. Acad. Sci. USA. 92:10267-10271, describe knocking out the DHFR-TSgene to produce a safe, live Leishmania vaccine, which publication isincorporated by reference. By disrupting the DHFR-TS gene in Neospora,auxotrophic mutants will be created that require thymidine for continuedgrowth, that exhibit attenuated pathogenicity, and that are capable oftriggering an immune response in a mammal that protects againstneosporosis when administered as a live vaccine.

Recombinant DNA techniques for gene replacement or gene knockout areknown in the art and include, but are not limited to, those that takeadvantage of homologous recombination. For example, cells of apathogenic strain of Neospora may be transformed or transfected with avector, such as a plasmid, comprising homologous nucleotide sequencesthat normally flank, or are located with, for example, an essentialmetabolic gene, preferably a single copy gene, in a pathogenic strain ofNeospora. Between or within the homologous nucleotide sequences, thevector may further comprise a nucleotide sequence that corresponds tothe nucleotide sequence in the pathogenic strain but which is defectiveas a result, for example, of a “non-silent” change or deletion in one ormore nucleotides compared to the sequence from the pathogenic strain.Transformation of a cell of the pathogenic strain with the vector isfollowed by integration of the defective gene sequence into the Neosporagenome, which also serves to replace the original or “wild-type”sequence. Thus, the targeted gene is disabled in the transformed cell.Transformed cells may then be screened for those cells that exhibit anattenuated pathogenicity. Transformed cells exhibiting attenuatedpathogenicity may then be screened again for those cells that arecapable of triggering an immune response in a mammal that protectsagainst neosporosis when administered as a live vaccine.

To aid in the selection of transformants, the vector may be engineeredto further comprise a coding sequence for a reporter gene product orother selectable marker. Reporter genes which may be useful in theinvention are well-known in the art and include, for example, the geneencoding chloramphenicol acetyl transferase (CAT), or the gene encodingluciferase. A further non-limiting example of a reporter gene is asequence encoding E. coli β-galactosidase, which can be inserted intothe vector and used to confirm transformants by detecting enzymaticactivity through conversion of a substrate such as, for example,red-β-D-galactopyranoside, to a colored product. Seeber and Boothroyd,1996, Gene, 169:39-45, used this reporter enzyme to detect transformantsin Toxoplasma gondii, which publication is incorporated herein byreference.

Coding sequences that encode selectable markers which may be useful inthe invention are also well-known in the art, and include those thatencode gene products conferring resistance to antibiotics oranti-metabolites, or that supply an auxotrophic requirement. Examples ofsuch sequences include those that confer resistance to hygromycin, or toneomycin, or to phleomycin. An example of the use of an antibioticresistance marker in a different pathogen is presented by Messina etal., 1995, Gene, 165:213-217, which describes the use of a resistancemarker for phleomycin to construct stable transformants in T. gondii,and which publication is incorporated herein by reference.

Any coding sequence for a reporter gene product or selectable markershould preferably be inserted into the vector in operative associationwith a regulatory element coding sequence. As used herein, a “regulatoryelement” includes, but is not limited to, inducible and non-induciblepromoters, enhancers, operators and other elements known in the art thatserve to drive and/or regulate expression. Also, as used herein, a DNAcoding sequence is in “operative association” with one or moreregulatory elements where the regulatory elements effectively regulateand allow for the transcription of the DNA coding sequence and/or thetranslation of the corresponding mRNA. For example, vectors forexpression of the selectable marker ble in Neospora cells may beconstructed by flanking the ble open reading frame (ORF) with regulatoryregions from genes of Neospora or from the closely related Apicomplexa,Toxoplasma. The 5′ flanking regions from different single copy Neosporaor Toxoplasma genes may be used to express ble. Examples of single copyToxoplasma genes are: (i) SAG1. encoding the major tachyzoite surfaceantigen, p30 (Burg et al., 1988, J. Immunol. 141:3584-3591); (ii) GRA1,encoding a secretory protein, p23 (Cesbron-Delauw et al., 1989. Proc.Natl. Acad. Sci. 86:7537-7541): and (iii) GRA2, encoding a secretoryprotein, p28 (Mercier et al, 1993, Mol. Biochem. Parasitol. 58:71-82).Examples of single copy Neospora genes include: (i) homologs of theToxoplasma SAG1, GRA1 and GRA2 genes, identified using standard PCRmethods based, for example, on the published Toxoplasma sequences; (ii)the gene encoding the major cell surface protein, NC-p43, of N. caninumtachyzoites (Hemphill. 1996, Infect. Immun. 64:4279-4287); and (iii)genes encoding the immunodominant 17-, 29-, 30- and 37 kDaexcretory/secretory proteins (Bjerkas et al., 1994, Clin. Diag. Lab.Immun. 1:214-221). The 3′ flanking region from the SAG1 gene may be usedto provide a polyadenylation sequence. The vector backbone for insertionof the 5′ promoter, ble gene, and 3′ polyadenylation sequences may beany standard, commercially available plasmid, such as pBLUESCRIPT™(Stratagene).

Once an appropriate vector is constructed, it is used to transform ortransfect one or more cells from a parental strain of Neospora. Thevector may be introduced into the cells in accordance with knowntechniques, including but not limited to electroporation,microinjection, viral transfection, liposome-mediated transfection,microprojectile bombardment, etc.

Once the vector is introduced into the Neospora cells, the presence,integration and maintenance of the introduced coding sequence into thehost cell genome, or episomally, can be confirmed and monitored bystandard techniques including, but not limited to, Southernhybridization analysis; PCR analysis, including reversetranscriptase-PCR (RT-PCR); immunological or calorimetric assay for theexpected protein product; detecting the presence or absence of a markergene function, such as appearance of a novel auxotrophy; or by detectingan attenuation in pathogenicity.

Examples of vector construction, transformation, selection oftransformants, host cell expression, etc., as applied specifically topathogenic protozoans, are described in the following publications,which are incorporated herein by reference: Seeber and Boothroyd, 1996,above; Titus et al., 1995. above: Messina et al., 1995, Gene,165:213-217; Sibley et al. 1994, Proc. Natl. Acad. Sci. USA,91:5508-5512; Donald and Roos, 1994, Mol. Biochem. Parasitol.,63:243-253; Kim et al., 1993, Science, 262:911-914; Ryan et al, 1993,Proc. Natl. Acad. Sci. USA, 90:8609-8613; Soldati and Boothroyd, 1993,Science, 260:349-352; Eid and Soliner-Webb, 1991, Proc. Natl. Acad. Sci.USA, 88:2118-2121; LeBowitz et al., 1990, Proc. Natl. Acad. Sci. USA,87:9736-9740; Lee and Van der Ploeg, 1990, Science, 250:1583-1586:Asbroek et al., 1990, Nature, 348:174-175; Cruz and Beverley, 1990,Nature, 348:171-173; and Laban et al., Nature, 343:572-574.

General techniques of genetic recombination, including vectorconstruction, transformation, selection of transformants, host cellexpression, etc., are further described in Maniatis et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Ausubel et al., 1989. Current Protocolsin Molecular Biology, Greene Publishing Associates & Wiley Interscience,N.Y.; Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., SanDiego, Calif.; and Erlich (ed), 1992, PCR Technology, Oxford UniversityPress, New York, which are incorporated herein by reference.

After the attenuation step, cells that exhibit one or more indicators ofattenuated pathogenicity are selected from the culture and clonallypropagated after limiting dilution. Examples of such indicators include,but are not limited to, appearance of a novel temperature-sensitivity ora novel auxotrophy in vitro, or a reduction in a virulence attributesuch as infectivity or severity or rate of progression of one or moresymptoms or conditions in a mammal after administration of cells of thestrain as compared to infection with the parent strain, among others. Aparticular, non-limiting example of a temperature-sensitivity that isuseful in practicing the invention is one in which cells of theattenuated strain will grow at 32° C., but not at 37° C. Such atemperature-sensitive strain will cause the lysis of infected host cellsat 32° C., resulting in the appearance of lesions or plaques in a hostcell monolayer. When grown at 37° C., the attenuated strain will notreplicate sufficiently and will thus fail to produce plaques in hostcell monolayers.

An attenuated strain of Neospora may be derived from any pathogenicstrain of any species of the genus including, but not limited to, N.caninum. A non-limiting example of a particular pathogenic strain of N.caninum from which an attenuated strain may usefully be derived isstrain NC-1 which is present in infected MARC145 monkey kidney cellsfrom the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 USA (ATCC Accession No. CRL-12231). Strain NC-1 isalso described in Dubey et al., 1988, J. Am. Vet. Med. Assoc.193:1259-63, which publication is incorporated herein by reference.Alternatively, pathogenic strains of Neospora may be obtained fromtissues, organs or body fluids of infected animals exhibiting clinicalsymptoms of neosporosis using standard isolation techniques described,for example, in the publications reviewed above. A non-limiting exampleof a live, attenuated strain derived from the NC-1 strain of N. caninumis NCTS-8 which is present in infected MARC145 monkey kidney cells fromthe ATCC (ATCC Accession No. CRL-12230).

Both parental strains and attenuated strains of Neospora may be culturedin vitro by infecting any receptive cell line, preferably a mammaliancell line, with tachyzoites of the strain according to known techniquesdescribed in the art. Mammalian cell lines in which tachyzoites ofNeospora can be cultured include, for example, human foreskinfibroblasts (Lindsay et al., 1993, Am. J. Vet. Res. 54:103-106); bovinecardiopulmonary aortic endothelial cells (Marsh et al., 1995, above);and bovine monocytes (Lindsay and Dubey, 1989, above), among others. Forexample, tachyzoites of N. caninum may be cultured in monolayers of Hs68human foreskin fibroblast cells (ATCC Accession No. CRL-1635) (Lindsayet al., 1993, above). Bradyzoites may be similarly cultured andmanipulated.

Mammalian cell cultures can be grown, and cell cultures infected withNeospora can be maintained, in any one of several culture mediadescribed in the art. For example, stationary monolayer cultures ofbovine cardiopulmonary aortic endothelial cells infected withtachyzoites of N. caninum may be grown in Dulbecco's Minimum EssentialMedium (DMEM: Gibco Laboratories, N.Y.), supplemented with 10% (v/v)heat-inactivated fetal bovine serum (FBS) or adult equine serum (ES), 2mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin (Conrad etal., 1993, above). Monolayers of Hs68 human foreskin fibroblast cellsmay be maintained in RPMI 1640 (Gibco) containing 2% (v/v) fetal bovineserum, 1.0 mM sodium pyruvate, 1×10⁴ U/ml penicillin, 1×10⁴ μg/mlstreptomycin, 5×10⁻² mM 2-mercaptoethanol and 0.3 mg/ml L-glutamine(maintenance medium). Monolayer cultures of Hs68 human foreskinfibroblast cells infected with Neospora may be maintained in identicalmedia, but in which the fetal bovine serum is increased to 10% (v/v)(growth medium). Attenuated strains of Neospora having novelauxotrophies will require appropriate modification to the culture mediumto support growth, as known in the art.

Neospora-infected monolayer cultures of mammalian cells are typicallymaintained under standard tissue culture conditions such as, forexample, at 37° C. and 5% CO₂. Tachyzoites are generally passaged touninfected monolayer cultures when 70-90% of the mammalian cells in theculture have become infected, which may be determined microscopicallyusing standard techniques. Tachyzoites may be collected from theinfected mammalian cell cultures by lysing the host cells using anystandard technique that allows the tachyzoites to retain viability, andcollecting the tachyzoites by filtration or by centrifugation, forexample.

Preparation and Use of Vaccines

The present invention provides vaccines against neosporosis, comprisingan immunologically effective amount of live cells of a strain derivedfrom a pathogenic parent strain of a species of Neospora, which cellsexhibit attenuated pathogenicity compared to those of the parent strainbut which are capable of triggering an immune response that protects themammal against neosporosis when administered as a live vaccine, and aveterinarily acceptable carrier.

The present invention further provides methods for preparing a vaccinethat protects a mammal against neosporosis, comprising modifying cellsfrom a pathogenic parent strain of a species of Neospora; selecting andclonally propagating those modified cells that exhibit attenuatedpathogenicity compared to cells of the parent strain but which arecapable of triggering an immune response in the mammal that protectsagainst neosporosis when administered in a live vaccine; and combiningan immunologically effective amount of the attenuated cells with aveterinarily acceptable carrier in a form suitable for administration asa live vaccine to the mammal.

The present invention further provides methods of vaccinating a mammalagainst neosporosis, comprising administering to the mammal animmunologically effective amount of a vaccine comprising live cells of astrain derived from a pathogenic parent strain of a species of Neospora,which cells exhibit attenuated pathogenicity compared to those of theparent strain but which are capable of triggering an immune responsethat protects the mammal against neosporosis when administered as a livevaccine, and a veterinarily acceptable carrier.

The vaccine of the invention comprises live cells of an attenuatedstrain of Neospora, either as the sole antigenic component or incombination with one or more other antigens that trigger an immuneresponse that protects a mammal against a disease or pathologicalcondition which may or may not be related to neosporosis. Thus, thepresent invention further provides combination vaccines, comprising animmunologically effective amount of live cells of a strain derived froma pathogenic parent strain of a species of Neospora, which cells exhibitattenuated pathogenicity compared to those of the parent strain butwhich are capable of triggering an immune response that protects themammal against neosporosis when administered as a live vaccine; one ormore other antigens that trigger an immune response that protects themammal against a disease or a pathological condition; and a veterinarilyacceptable carrier. The combination vaccines may further comprise one ormore other components including, for example, an adjuvant.

The vaccine is conventionally administered parenterally, for example,either by subcutaneous or intramuscular injection. However, the vaccinemay also be administered by intraperitoneal or intravenous injection, orby other routes, including orally, intransally, rectally or vaginally,and where the vaccine is so administered, a veterinarily acceptablecarrier is appropriately selected. The vaccine may simply compriseattenuated cells in culture fluid, which are administered directly tothe mammal. Alternatively, the vaccine may comprise attenuated cellscombined with a veterinarily or pharmaceutically acceptable carrierselected from those known in the art based on the route ofadministration and its ability to maintain cell viability. Non-limitingexamples of such carriers include water, saline, buffered vehicles andthe like. Suitable other vaccine vehicles and additives are known, orwill be apparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

The vaccine may further comprise one or more other components such as animmunomodulatory agent including, for example, interleukin-1, or anotherimmuno-enhancing substance such as a veterinarily acceptable adjuvant.Non-limiting examples of adjuvants include Freund's complete andincomplete adjuvants, mineral gels including, for example, aluminumhydroxide, and oil-in-water or water-in-oil formulations.Immunomodulatory agents are selected based on their ability to maintainboth viability of the attenuated Neospora cells and ability of the cellsto trigger a protective immune response in the vaccinated mammal.

Non-limiting examples of oil-in-water formulations that are useful asadjuvants in the vaccines of the invention include emulsions 1-3, asfollows. Emulsion 1 consists of: (a) ME6201 (5% v/v squalene. 0.1% v/vvitamin E, and 0.8% v/v Tween™ 80 dispersant); (b) Quil A (QA) saponinpreparation (Superfos) (200 μg/ml); and (c) cholesterol (chol.) (100μg/ml). Emulsion 2 consists of: (a) ME6201: and (b) Avridine lipoidalamine (1 mg/ml). Emulsion 3 consists of ME6210 (5% v/v squalene, 1.0%v/v vitamin E, and 0.8% v/v Tween™ 80 dispersant).

An effective dosage may be determined by conventional means, startingwith a low dose of attenuated cells and then increasing the dosage whilemonitoring the effects, and systematically varying the dosage as well.Numerous factors may be taken into consideration when determining anoptimal dosage per animal. Primary among these is the species, the sizeof the animal, the age of the animal, the general condition of theanimal, the presence of other drugs in the animal, the virulence of aparticular strain of Neospora against which the animal is beingvaccinated, and the like. The actual dose would preferably be chosenafter consideration of the results of other animal studies.

Vaccine regimens are selected also based on the above-described factors.Animals may be vaccinated at any time, including just prior to or at thetime of breeding. Supplemental administrations, or boosters, may berequired for full protection. One method of detecting whether adequateimmune protection has been achieved is to determine seroconversion andantibody titers in the animal after vaccination. Thus, the vaccine ofthe invention may be administered at any time during the life of aparticular animal to be vaccinated, depending upon several factors,including, for example, the timing of an outbreak of neosporosis amongother animals. etc. The vaccine may be administered to animals ofweaning age or younger, or to more mature animals, for example, as apre-breeding vaccine to protect against Neospora-related congenitaldisease or abortion. Effective vaccination may require only a primaryvaccination, or a primary vaccination with one or more boostervaccinations. Booster vaccinations may be administered at any time afterprimary vaccination depending, for example, on the immune response afterprimary vaccination, the severity of the outbreak, the virulence of thestrain of Neospora causing the outbreak, the health of the vaccinate,etc. The timing of vaccination and the number of boosters, if any, willpreferably be determined by a veterinarian based on analysis of allrelevant factors, some of which are described above.

The Neospora cells used in the vaccine are preferably tachyzoites, butmay instead be bradyzoites, or oocysts, or some combination thereof. Theconcentration of attenuated cells in the vaccine preferably ranges fromabout 1×10³/ml to about 1×10⁸/ml, and more preferably from about2×10⁶/ml to about 2×10⁷/ml. A suitable dosage size ranges from about 0.5ml to about 1.0 ml. Generally, on a per-dose basis, the number ofattenuated cells administered to an animal is preferably from about2×10⁴ to about 2×10⁸; more preferably from about 2×10⁵ to about 2×10⁷;and most preferably from about 1×10⁶ to about 1×10⁷.

The vaccine of the invention protects a mammal against infection ordisease caused by Neospora. The vaccine is useful to protect bothpregnant and non-pregnant mammals including, but not limited to, bovine,ovine, caprine, canine and equine species, against infection, clinicaldisease or abortion resulting from neosporosis. The term “protection” isused broadly and is not limited to absolute prevention of infection byNeospora, but includes a reduction in infectivity, or in the severity ofa disease or condition resulting from infection, including a detectablereduction in one or more of the pathological effects or symptomsresulting from infection, or a detectable reduction in the rate ofprogression of one or more of such pathological effects or symptoms. Thevaccine of the invention is also safe, i.e., it does not cause diseaseor significant side effects in the vaccinated mammal.

The following examples are offered to further illustrate, but not limit,the compositions and methods of the invention.

EXAMPLE 1 Establishment of Temperature-Sensitive Strains of N. caninumand Analysis of Pathogenicity in BALB/c Mice

The objective of this study was to establish temperature-sensitivestrains of N. caninum (NCTS), and to test the pathogenicity of thesestrains by analyzing serum antibody response, issue cyst and brainlesion production, and the development of clinical symptoms in BALB/cmice, which are known to be highly susceptible to neosporosis.

Materials and Methods

Tachyzoites of the NC-1 strain of N. caninum (Dubey et al., 1988, above)were cloned twice by limiting dilution and maintained at 37° C., asdescribed (Lindsay and Dubey, 1989, above). The tachyzoites werepropagated in 25 cm² flasks and cloned in 96-well plates containingmonolayers of Hs68 human foreskin fibroblast cells (ATCC Accession No.CRL-1635) (Lindsay et al., 1993, above). The Hs68 cells were previouslygrown in RPMI 1640 containing 2% (v/v) fetal bovine serum, 1.0 mM sodiumpyruvate, 1×10⁴ U/ml penicillin, 1×10⁴ μg/ml streptomycin, 5×10⁴ mM2-mercaptoethanol and 0.3 mg/ml L-glutamine (maintenance medium).Infected cell culture monolayers were maintained in identical medium,but in which fetal bovine serum was increased to 10% (v/v) (growthmedium). A clone was isolated and designated in the laboratory as theNC-1-2C line, but is referred to hereinafter simply as the NC-1 strain.

Tachyzoites of the NC-1 strain were mutagenized by exposure to 0.5 μMN-methyl-N′-nitro-N-nitrosoguanidine (Sigma) in growth medium for 24 hr,and then grown at 32.5° C. for 3 mos in Hs68 cells in maintenancemedium, after which tachyzoites were cloned by limiting dilution. Twelveclones were initially isolated. Three clones, designated as NCTS-4,NCTS-8, and NCTS-12 (NCTS=N. caninum, temperature-sensitive), wereselected for further study after being maintained in Hs68 cells incontinuous culture at 32.5° C. for >8 mos in maintenance medium.

Serological testing of mice was conducted as follows. An indirectimmunofluorescent antibody test (IFAT) (Dubey et al., 1988, above) wasused to analyze sera from mice for the presence of antibodies directedagainst N. caninum. Mouse sera were obtained immediately prior tochallenge inoculation in vaccination studies. Serum was also obtainedfrom all mice that survived pathogenicity experiments. Sera wereexamined at doubling dilutions beginning at 1:50 and endpoint titrated.Tachyzoites were used as antigen. Positive samples exhibited a completetachyzoite surface fluorescence. Negative samples exhibited nofluorescence or only anterior end fluorescence.

The presence of lesions in the brains of (nice challenged with differentstrains of N. caninum was determined as follows. The brain from eachmouse was removed at necropsy, and a first half was fixed in 10% v/vneutral buffered formalin solution for histopathological examination.Tissue sections were prepared using routine histological techniques, andstained with hematoxylin and eosin to detect the presence of lesionsmicroscopically.

Lesion scoring of hematoxylin- and eosin-stained brain tissue sectionswas conducted according to the criteria described in Lindsay et al.,1995, J. Parasitol. 81:313-315. Number of inflammatory/necrotic fociScore No foci 1 1-5 foci 2 6-10 foci 3 >10 foci 4

Average size of foci Score None 1 100-200 μm 2 200-500 μm 3 >500 μm 4

Severity of lesions Score No lesions 1 slight 2 mild 3 moderate 4 marked5

A mean lesion score was obtained using the three values listed above. Anormal, non-infected mouse brain would have a mean lesion score of 3.0.Mean lesion scores were evaluated using a Kruskal-Wallis nonparameterictest, and distribution free multiple comparison methods. The number ofmice in each group with lesions was examined using Fisher's exact test.Statistical significance was established at a cutoff of P<0.05.

Sections of brain from each mouse were also examined using a murinemonoclonal antibody, 6G7, specific for N. caninum, in conjunction withan avidin-biotin peroxidase complex (ABC) immunohistochemical test, todetect N. caninum stages (Cole et al., 1993, J. Vet. Diag. Invest.5:574-589).

The second half of each mouse brain was digested in acid-pepsin and usedto inoculate mammalian cell cultures to detect the presence of tissuecysts of N. caninum. To digest, the second half of the mouse brain wasplaced in 3 ml of Hank's balanced salt solution (HBSS) and passed twicethrough a syringe with a 23 gauge needle. Three ml of acid-pepsinsolution (0.52 g pepsin, 0.50 g NaCl, 98.6 ml dH₂O, 1.4 ml conc. HCl, pH0.8), was added to the homogenate and incubated for 10 min at 37° C. ina water bath. The acid pepsin solution was removed by centrifugation andthe pellet, representing the entire second brain half, was inoculatedinto a single 25 cm² tissue culture flask containing a monolayer of Hs68human foreskin fibroblast cells cultured as described above. After 30min, the inoculum was removed and the Hs68 cell monolayer was washed andincubated in fresh maintenance medium as above. Cell cultures were thenexamined for 30 days to detect the presence of N. caninum (Lindsay andDubey, 1989, above).

The pathogenicity of NC-1 and the three selected NCTS strains, i.e.,NCTS-4, NCTS-8, and NCTS-12. of N. caninum were determined as follows.BALB/c mice (8 wk, female) (Harlan Sprague Dawley (HSD)) were inoculatedsubcutaneously with HBSS (control), or with 5×10⁵ tachyzoites of eitherthe NC-1, NCTS-4, NCTS-8, or NCTS-12 strain of N. caninum in HBSS (0.5to 1.2 ml total volume). Surviving mice were examined at necropsy 42 or56 days post-inoculation (PI) (see Table 1). Serum was collected fromsurviving mice at necropsy. Brains of these mice were examined forlesion scoring and immunohistology, and one half of each brain was usedfor acid-pepsin digestion to determine the presence of tissue cysts, asdescribed above. TABLE 1 Results of inoculating BALB/c mice withtachyzoites of NCTS-4, NCTS-8, NCTS-12 or NC-1 strains of N. caninum.Treatment N. caninum Group No. Mice Strain Result 1 4 HBSS No mortality.(control) 2 5 NCTS-4 No mortality. 3 5 NCTS-4 No mortality. 4 5 NCTS-8No mortality. 5 5 NCTS-8 No mortality. 6 5 NCTS-12 No mortality. 7 5NCTS-12 No mortality. 8 5 NC-1 Clinical neosporosis developed at about16 days PI; mice were less active than in groups 1-7; with rough haircoats. Two mice were euthanized because of neosporosis at 34 days PI;one additional mouse died 39 days PI. 9 5 NC-1 Clinical signs same as ingroup 8; mice found dead on days 26, 30, 32 and 41 PI.

TABLE 2 Mean lesion scores of BALB/c mice inoculated with differentstrains of N. caninum. No. mice with lesions/No. examined/No. TreatmentGroup survived Mean Lesion Score 1 0/4/4 3.00 (HBSS-control) 2 + 34/10/10^(a) 4.80 (NCTS-4) 4 + 5 6/10/10^(a) 5.50 (NCTS-8) 6 + 75/10/10^(a) 4.90 (NCTS-12) 8 + 9 8/8/3^(a,b) 9.38^(a,b) (NC-1)^(a)= significantly different from control (HBSS) (P < 0.05).^(b)= significantly different from NCTS-4, NCTS-8, and NCTS-12 (P <0.05).

TABLE 3 Reciprocal antibody titers in mouse serum. Reciprocal AntibodyTiter Treatment Group <50 800 1,600 3,200 6,400 1 (HBSS-control) 2 — — —— 42 Days PI 1 (HBSS) 2 — — — — 56 Days PI 2 (NCTS-4) — 1 4 — — 42 DaysPI 3 (NCTS-4) — — 5 — — 56 Days PI 4 (NCTS-8) — — 3 2 — 42 Days PI 5(NCTS-8) — — 3 2 — 56 Days PI 6 (NCTS-12) — — 3 2 — 42 Days PI 7(NCTS-12) — — 4 1 — 56 Days PI 8 (NC-1) — — 2 — — 56 Days PI 9 (NC-1) —— 1 — — 56 Days PI

Results

Clinical neosporosis and mortality occurred only in BALB/c miceinoculated with the NC-1 strain (Table 1). Only three out of 10 BALB/cmice survived the NC-1 strain infection. The NCTS-4. NCTS-8, and NCTS-12strains did not cause mortality in BALB/c mice.

Mean lesion scores are presented in Table 2. Lesions were found inbrains of some mice inoculated with the NCTS strains, but mean lesionscores were not statistically significant when compared to the control(HBSS). A significant difference in mean lesion scores and numbers ofmice with lesions was found when mice inoculated with the NC-1 strainwere compared to mice inoculated with HBSS (control) or with any of theNCTS strains of N. caninum.

Serum antibody titers are presented in Table 3. Significant IFAT titers(≧400) were detected in all mice challenged (30/30) with NCTS strains.This indicates that NCTS strains are capable of stimulating a B-cellresponse in an immunologically intact, but genetically susceptible,animal. IFAT titers in NCTS-challenged mice were equal to, or in somecases higher than, those in NC-1 challenged mice.

Tissue cysts were not detected in any of the brain halves examined usingthe acid-pepsin digestion technique described above. This indicates thattissue cysts, if present, are few in number in mice inoculated with theNCTS or NC-1 strains of N. caninum.

EXAMPLE 2 Analysis of Pathogenicity of NCTS Strains of N. caninum inHSD:ICR Outbred Mice

The objective of this study was to determine the pathogenicity of NCTSstrains of N. caninum in HSD:ICR outbred mice, which are immunocompetentand more resistant to Neospora than inbred BALB/c mice.

Materials and Methods

HSD:ICR mice (4 wk, female) were used in this experiment All HSD:ICRmice, except for controls, were inoculated with 5×10⁵ tachyzoites of theappropriate strain of N. caninum in HBSS (total volume 0.5 to 12 ml).Control mice were inoculated with HBSS only. All surviving mice weresacrificed 56 days PI. Serum was collected for IFAT testing. Brains fromthese mice were collected and a first half was used for lesion scoringand immunohistology, as above. The second half of each brain was used inthe acid-pepsin digestion to detect tissue cysts, as described above.

Results

None of the N. caninum strains tested, including NC-1, caused mortalityin HSD:ICR mice (Table 4), and no significant differences were observedin numbers of mice with lesions or in the mean lesions scores comparedto non-challenged controls (Table 5). No brain tissue cysts wereobserved in histological or ABC-stained sections. No parasites wereisolated in cell cultures. TABLE 4 Results of inoculating HSD:ICR micewith tachyzoites of NC-1, NCTS-4, NCTS-8 or NCTS-12 strains of N.caninum. Treatment N. caninum Group No. Mice Strain Result 10 5 HBSS Nomortality. (control) 11 5 NC-1 No mortality. 13 5 NCTS-4 No mortality.14 5 NCTS-8 No mortality. 15 5 NCTS-12 No mortality.

TABLE 5 Mean lesion scores of HSD:ICR mice inoculated with variousstrains of N. caninum. No. mice with lesions/ No. examined/No. TreatmentGroup survived Mean Lesion Score^(a) 10 0/5/5 3.0 (HBSS-control) 113/5/5 6.0 (NC-1) 13 0/5/5 3.0 (NCTS-4) 14 1/5/5 4.0 (NCTS-8) 15 0/5/53.0 (NCTS-12)^(a)= No significant differences were observed between treatment groups.

TABLE 6 Reciprocal antibody titers in mouse serum.^(a) TreatmentReciprocal Antibody Titer Group <50 400 800 1,600 3,200 6,400 10(HBSS-control) 5 — — — — — 11 (NC-1) — — — 2 3 — 13 (NCTS-4) — 1 3 1 — —14 (NCTS-8) 1 — 2 1 1 — 15 (NCTS-12) 1 — 3 1 — —^(a)All titers were determined at day 56 PI.

Antibody titers of surviving mice are presented in Table 6. SignificantIFAT titers (≧400) were detected in the majority of mice (13/15)challenged with the NCTS strains, indicating that these strains arecapable of inducing a B-cell response in immunologically intact butgenetically resistant, animals.

Tissue cysts were not detected in the portion of brain examinedhistologically, immunohistologically, or by using the acid-pepsindigestion procedure. These results confirm those presented in Example 1,above, using BALB/c mice.

Example 3 Analysis of Pathogenicity of NCTS Strains of N. caninum inImmunosuppressed HSD:ICR Mice

A first objective of this study was to determine the pathogenicity ofNCTS strains of N. caninum in immunosuppressed HSD:ICR mice. A secondobjective of this study was to determine if reversion to pathogenicityoccurs after in vitro passage of NCTS strains of N. caninum at 37° C.

Materials and Methods

HSD:ICR mice (4 wk, female) were immunosuppressed by intramuscularadministration of 2 mg m thylprednisolone acetate (MPA)(Upjohn-Pharmacia) on days −7, 0, and 7 PI. See Lindsay and Dubey. 1989,J. Parasitology 75:772-779.

The immunosuppressed HSD:ICR mice were inoculated on day 0 with 2×10⁵tachyzoites of either the NC-1, NCTS-4, NCTS-8, or NCTS-12 strains, orone of the potential reversion controls, designated NCTS-4-37,NCTS-8-37, or NCTS-12-37, in HBSS (total volume 0.5 to 1.2 ml). The micewere subsequently examined serologically, histologically, andclinically, as described above.

NCTS-4, NCTS-8, and NCTS-12 clones were examined for reversion topathogenicity by growth at 37° C. for 88 days (25 cell culture passes),followed by inoculation in immunosuppressed HSD:ICR mice (potentialreversion strains are designated as NCTS-4-37. NCTS-8-37, NCTS-12-37).

Results

The NC-1, NCTS-4-37, and NCTS-12-37 strains of N. caninum caused 100%mortality in immunosuppressed HSD:ICR mice (Table 7). The NCTS-4,NCTS-8, NCTS-12. and NCTS-8-37 strains were less pathogenic towardimmunosuppressed HSD:ICR mice and caused only from 0 to 20% mortality.Mean lesion scores are presented in Table 8. No tachyzoites weredetected in acid-pepsin digests from any mice examined at necropsy 56days PI.

Antibody titers of surviving mice are presented in Table 9. SignificantIFAT titers (≧800) were detected in NCTS-4, -8, and -12. and NCTS-8-37mice at day 56 post-challenge, indicating that these strains are capableof stimulating a B-cell response in an immunosuppressed, but geneticallyresistant, animal. TABLE 7 Results of inoculating immunosuppressedHSD:ICR mice with tachyzoites of NC-1, NCTS-4, NCTS-8, NCTS-12, orpotential reversion strains of N. caninum. Treatment No. N. caninumGroup Mice Strain Result 16 5 HBSS No mortality. (control) 17 5 NC-1 3mice died or were euthanized* on day 20, and one mouse died on each ofdays 22 and 23 PI. 18 5 NCTS-4 No mortality. 19 5 NCTS-8 1 mouseeuthanized 20 days PI.* 20 5 NCTS-12 1 mouse died 22 days PI. 21 5NCTS-4-37 All mice died or were euthanized at 13, 14 (2), 16 and 21 daysPI.* 22 5 NCTS-8-37 1 mouse died 29 days PI. 23 5 NCTS-12-37 1 mousedied and 4 were euthanized 14 days PI.**= Mice that were moribund due to clinical encephalitic neosporosis wereeuthanized for humane reasons.

TABLE 8 Mean lesion scores of immunosuppressed HSD:ICR mice inoculatedwith various strains of N. caninum. No. mice with Treatment lesions/No.Group examined/No. survived Mean Lesion Score 16 0/5/5 3.0(HBSS-control) 17 4/4/0^(a) 9.8^(a) (NC-1) 18 1/5/5 3.6 (NCTS-4) 192/5/4 5.2 (NCTS-8) 20 3/5/4 6.2 (NCTS-12) 21 3/3/0^(a) 8.7 (NCTS-4-37)22 5/5/4^(a) 8.2 (NCTS-8-37) 23 4/4/0^(a) 9.5^(a) (NCTS-12-37)^(a)= significantly different from control (Group 16) (P < 0.05).

TABLE 9 Reciprocal antibody titers in mouse serum.^(a) TreatmentReciprocal Antibody Titer Group <50 100 800 1,600 3,200 6,400 12,800 16(HBSS- 5 — — — — — — control) 18 (NCTS- — 1 1 1 1 1 — 4) 19 (NCTS- — — —2 1 1 — 8) 20 (NCTS- — — — 1 2 1 — 12) 22 (NCTS- — — — — 1 2 1 8-37)^(a)All titers were determined at day 56 PI.

The NCTS strains were less pathogenic in immunosuppressed HSD:ICR micethan the NC-1, NCTS-4-37 and NCTS-12-37 strains. The relatively highsurvival rate (4/5) of mice inoculated with the NCTS-8-37 reversionstrain compared with the 100% mortality rate of mice inoculated with theNCTS-4-37 or NCTS-12-37 reversion strains indicates that the NCTS-8-37reversion strain retained attenuated pathogenicity following serialpassage at 37° C. Based on this demonstrated retention of attenuatedpathogenicity, the NCTS8 strain of N. caninum was selected as apotential vaccine candidate and used in further studies, as describedbelow.

EXAMPLE 4 Vaccination Of BALB/c Mice Against N. caninum-InducedEncephalitis

A first objective of this study was to determine if vaccination with alive, temperature-sensitive strain of N. caninum can provide protectionagainst disease caused by subsequent challenge with a pathogenic strain.e.g., NC-1, of N. caninum. A second objective of this study was todetermine the level of protection provided by vaccination of BALB/c micewith killed (frozen) NCTS-8 tachyzoites that were subsequentlychallenged with the NC-1 strain of N. caninum.

Materials and Methods

BALB/c mice (9 wk, female) were vaccinated by subcutaneous injectionwith either HBSS (control) (0.5 ml), or 5×10⁵ living tachyzoites of theNCTS-8 strain in HBSS (0.5 ml), or 2×10⁶ killed (frozen) tachyzoites inHBSS (0.5 ml) (Table 10). The vaccinates were boosted 21 days PI withthe same material as in the primary injection. The mice were thenchallenged by subcutaneous administration of 1×10⁶ tachyzoites of theNC-1 strain of N. caninum in HBSS, or with HBSS alone (control) (totalvolume 0.5 ml), 14 days after the booster.

IFAT was used to test sera from mice that survived the experiment forthe presence of antibodies against N. caninum, as described above. Thebrain from each mouse was removed at necropsy. The first half was usedfor histopathology and immunohistology, and the second half was used foracid-pepsin digestion, as described above. Viability of tachyzoitesafter freezing was determined by inoculating monolayer cultures of Hs68human foreskin fibroblast cells, as described above.

Results

None of the mice in any test group died after primary or boostervaccination. Two of 10 mice vaccinated with HBSS (controls), and 3 of 10mice vaccinated with killed NCTS-8 tachyzoites, died after beingchallenged with NC-1 tachyzoites. Mean lesion scores of mice thatsurvived are presented in Table 11.

Reciprocal antibody titers of surviving mice are presented in Table 12.Significant IFAT titers (≧800) were detected in mice vaccinated withlive NCTS-8 tachyzoites following the booster, confirming previousresults described above. In contrast, no significant IFAT titers werepresent in any mice vaccinated with killed NCTS-8 tachyzoites prior tochallenge, demonstrating that killed tachyzoites fail to induce asignificant antibody response in a genetically susceptible host.

N. caninum tachyzoites were isolated from cell cultures inoculated withacid-pepsin-digested brain tissue from two sham-vaccinated, challengedmice (group 24, mice nos. 1 and 2), and from one mouse vaccinated withkilled, NCTS-8 tachyzoites (group 31, mouse no. 5). No tachyzoites wereisolated from cultures inoculated with brain tissue from any other mice.TABLE 10 Protocol for vaccination and challenge of BALB/c mice.Treatment Vaccination & Group No. Mice Booster Challenge 24 5 HBSS NC-125 5 HBSS NC-1 26 5 NCTS-8 (Live) HBSS 27 5 NCTS-8 (Live) HBSS 28 5NCTS-8 (Live) NC-1 29 5 NCTS-8 (Live) NC-1 30 5 NCTS-8 (Killed) NC-1 315 NCTS-8 (Killed) NC-1

TABLE 11 Mean lesion scores of vaccinated, challenged BALB/c mice. No.mice with lesions/ Treatment No. examined/ Group No. survived Meanlesion score 24 + 25 8/8/8 9.3 26 + 27 1/10/10 3.4^(a,b) 28 + 29 1/10/103.3^(a,b) 30 + 31 6/7/7 7.7^(a)= significantly different from groups 24 + 25 (P < 0.05).^(b)= significantly different from groups 30 + 31 (P < 0.05).

TABLE 12 Reciprocal antibody titers in mouse serum pre- andpost-challenge. Pre- or Post Reciprocal Antibody Titer Treatment GroupChallenge <50 50 100 400 800 1,600 3,200 6,400 24 + 25 Pre- 10 — — — — —— — (HBSS-control) 24 + 25 Post- — — — — — 1 4 3 (HBSS-control) 26 + 27(NCTS-8, Pre- — — — 1 4 4 1 — live; HBSS) 26 + 27 (NCTS-8, Post- — — — —3 4 3 — live; HBSS) 28 + 29 (NCTS-8 Pre- — — — — 1 9 — — live; NC-1)28 + 29 (NCTS-8 Post- — — — — 1 8 1 — live; NC-1) 30 + 31 (NCTS-8 Pre- 6 2 2 — — — — — killed; NC-1) 30 + 31 (NCTS-8 Post- — — — 1 1 2 2 1killed; NC-1)

Mice vaccinated with live tachyzoites of the NCTS-8 strain of N. caninumdid not die or develop clinical disease symptoms. Mice vaccinated withlive tachyzoites of the NCTS-8 strain and subsequently challenged withtachyzoites of the NC-1 strain (Table 11. groups 28+29) had lesionscores that were almost identical to those of mice vaccinated with livetachyzoites of the NCTS-8 strain followed by administration of HBSS(Table 11, groups 26+27). This indicates that vaccination with livetachyzoites of the NCTS-8 strain provides substantial protection againstdisease caused by infection with the NC-1 strain of N. caninum.Vaccination of mice with killed tachyzoites of the NCTS-8 strain offeredlittle protection from neosporosis (Table 11, groups 30+31).

Example 6 Vaccination of BALB/c Mice With a Low Dose of the NCTS-8Strain of N. Caninum

The objective of this study was to determine if a low dose, i.e., 5×10⁴tachyzoites, of the NCTS-8 strain of N. caninum can provide protectionagainst a subsequent challenge infection.

Materials and Methods

BALB/c mice (7 wk, female) were vaccinated subcutaneously either withHBSS, or with 5×10⁴ tachyzoites from the NCTS-8 strain of N. caninum inHBSS (0.5 ml) (Table 13). This dose of tachyzoites is one-tenth theamount used in previous examples. The mice were boosted 21 days PI withthe same material as in the primary injection. The mice were thenchallenged 14 days after the booster with 1×10⁶ tachyzoites of the NC-1strain of N. caninum.

IFAT was used to test sera from mice that survived the experiment forantibodies against N. caninum, as described above. The brain from eachmouse was removed at necropsy. A first half was used for histopathologyand immunohistology, and the remaining second half was used for theacid-pepsin digestion, as described above.

Results

One control mouse (administered only HBSS) died after subsequentchallenge with tachyzoites of the NC-1 strain of N. caninum. None of themice vaccinated with the low dose of tachyzoites of the NCTS-8 strain ofN. caninum died after subsequent challenge with tachyzoites of the NC-1strain. Mean lesion scores and numbers of mice with lesions weresignificantly higher in control mice sham-vaccinated only with HBSS thanin mice vaccinated with a low dose of the NCTS-8 strain (Table 14).Reciprocal antibody titers are shown in Table 15. TABLE 13 Protocol forlow dose vaccination and challenge of BALB/c mice. Treatment Vaccination& Group No. Mice Booster Challenge 32 5 HBSS NC-1 36 5 NCTS-8 (5 × 10⁴NC-1 tachyzoites)

TABLE 14 Mean lesion scores in mice. No. surviving mice bearinglesions/No. Mean lesion Treatment Group mice surviving score 32(HBSS/NC-1) 4/4 10.25^(a) 36 (NCTS-8/NC-1) 2/5 4.50^(a)= significantly different from group 36 (P < 0.05).

TABLE 15 Reciprocal antibody titers in mouse serum prior to challengeinoculation. Reciprocal Antibody Titer Treatment Group <50 400 800 1,6003,200 6,400 32 (HBSS) 10 — — — — — anti-Neospora titer 36 (NCTS-8) — 3 43 — — anti-Neospora titer

Example 6 Efficacy of Vaccines With and Without Adjuvant

The objective of this study was to determine the effect of adding anadjuvant to a modified-live Neospora vaccine and the degree ofprotection obtained therefrom against neosporosis.

Materials and Methods

Previous in vitro results (data not shown) indicated that tachyzoites ofat least one modified-live N. caninum strain, i.e., NCTS-8, retainedpartial viability and infectivity in vitro when co-incubated with one ofseveral different oil-in-water formulations. Based on these in vitroresults, three specific formulations were selected for in vivoevaluation as adjuvants.

Groups of ten 15 wk female BALB/c mice were vaccinated subcutaneously(0.2 ml) on days 0 (primary vaccination) and 21 (booster) PI, eitherwith HBSS alone (control), or with 5×10⁵ tachyzoites of the NCTS-8strain of N. caninum in HBSS, or with 5×10⁵ tachyzoites of the NCTS-8strain of N. caninum in one of the following three oil-in-wateremulsions.

Emulsion 1 consisted of (a) ME6201 (5% v/v squalene, 0.1% v/v vitamin E,and 0.8% v/v Tween™ 80 dispersant); (b) Quil A saponin preparation (QA)(Superfos) (200 μg/ml); and (c) cholesterol (chol.) (100 μg/ml).

Emulsion 2 consisted of: (a) ME6201; and (b) Avridine lipoidal amine (1mg/ml).

Emulsion 3 consisted of ME6201 (5% v/v squalene, 1.0% v/v vitamin E, and0.8% v/v Tween™ 80 dispersant). TABLE 16 Protocol for testing vaccineformulations with and without adjuvant. Treatment Group Vaccine Emulsion38 HBSS none 39 NCTS-8 none 40 HBSS 1 41 NCTS-8 1 42 HBSS 2 43 NCTS-8 244 HBSS 3 45 NCTS-8 3

On day 35 PI, all mice were challenged by a subcutaneous administrationof 1×10⁸ tachyzoites of the NC-1 strain of N. caninum In HBSS (0.2 ml).Groups of 3 to 5 mice were sacrificed on days 49 and 63 PI forevaluation of vaccine efficacy.

Beginning on day 0, disease was assessed based on mortality, as well ason the appearance of hair coat ruffling, irregular movements, pelviclimb paralysis, and generalized weakness.

Histopathological analysis was carried out as follows. Lung samples wereobtained on day 49, fixed in 10% (v/v) neutral buffered formalin, andtissue was sectioned and stained using routine histological techniques.Hematoxylin- and eosin-stained lung sections were coded, and lesionswere scored in a blinded fashion without knowledge of treatment groups.Pneumonia lesions were scored using the following system: 0=none;1=mild; 2=moderate; 3=marked; 4=severe.

Results

Mice vaccinated with a formulation comprising tachyzoites from theNCTS-8 strain of N. caninum and an adjuvant had a significantly lowerincidence of mild pneumonia (33%) after challenge with the NC-1 strainof N. caninum than control mice vaccinated only with HBSS (56%)(P<0.01). None of the NCTS-8 vaccinated, non-challenged mice showed anysign of encephalitic disease or parasites when examined 9 weekspost-vaccination (data not shown), confirming that administration ofNCTS-8 does not produce clinical disease.

The results indicate that a formulation comprising attenuated, liveNeospora tachyzoites and an adjuvant is at least as effective and safefor use as a vaccine against neosporosis as the same formulation withoutan adjuvant (Table 17). TABLE 17 Histopathological analysis of mouselung tissue after administration of vaccine, with or without adjuvant,and challenge with the NC-1 strain of N. caninum. No. Mice with LungTreatment Lesions/No. Mice Treatment Group No. Examined Control Mice 383/5 (vaccinated with HBSS only) 40 3/4 42 2/4 44 2/5 Mice vaccinatedwith NCTS-8 39 0/4 without adjuvant Mice vaccinated with NCTS-8 with 411/4 adjuvant 43 0/4 45 3/4

Example 7 Protection of Pygmy Goats From Neosporosis

The objective of this study was to determine if vaccination of pygmygoats with an attenuated live strain of N. caninum can protect goalsagainst neosporosis. More specifically, the ability of a vaccinecomprising live tachyzoites of the NCTS-8 strain of N. caninum toprotect pygmy goat does against Neospora-induced abortion was tested.

Materials and Methods

Pygmy goat does of approximate age range 2-5 years were randomlyassigned to groups A-E (Table 18). The dose in each non-sham vaccineadministration (groups A-C) consisted of 4×10⁶ tachyzoites of theindicated strain. Following subcutaneous vaccination (1.0 ml/dose) onday 0 (primary) and day 21 (booster), the does were synchronized usingLUTALYSE™ prostaglandin preparation (Upjohn-Pharmacia) (10 mg/goat,intramuscular route) on days 28 and 39. The does were bred by naturalservice between days 52 and 56. Pregnant does were determined byultrasound and were between 41 and 55 days gestation at the time ofchallenge.

Does in groups A-D were challenged with 4×10⁶ tachyzoites of the NC-1strain of N. caninum in serum-free maintenance medium (0.45 ml)administered by jugular i.v. The does were then monitored by ultrasound,by temperature taken daily for 7 days post-challenge, and by visualobservation twice daily, and were bled once per week post-challenge.TABLE 18 Treatment groups of pregnant pygmy goat does. Vaccine ChallengeGroup No. Does Strain Adjuvant Strain A 4 NC-1 None NC-1 B 5 NCTS-8 NoneNC-1 C 4 NCTS-8 Emulsion 2

NC-1 D 6 Sham Emulsion 2 NC-1 E 2 Sham None None

Results

All goats vaccinated with either the rive NC-1 strain of N. caninum(group A) or the live, attenuated NCTS-8 strain of N. caninum (groups B,C) seroconverted and had measurable IFAT titers 10 days post-booster(Table 19). These data demonstrate that NC-1 and NCTS-8 are immunogenicin pregnant goats. The GMT for group A was numerically higher than forgroups B and C, suggesting enhanced replication in the host of the NC-1strain compared to the attenuated NCTS-8 strain.

Table 20 demonstrates the ability of a vaccine comprising live,attenuated tachyzoites of Neospora to protect pygmy goat does againstNeospora-induced abortion. All 4 goat does vaccinated with the live NC-1strain (A) experienced abortion after challenge with NC-1 (0%protection). Five of 6 goat does that were sham-vaccinated (D)experienced abortion after challenge with NC-1 (17% protection). Bycontrast, only 2 out of 5 goat does vaccinated with NCTS-8 (B) abortedafter challenge with NC-1 (60% protection), and only 2 out of 4 goatdoes vaccinated with NCTS-8 with adjuvant (C) aborted after challengewith NC-1 (50% protection).

These results demonstrate that pregnant pygmy goat does aresubstantially protected against Neospora-induced abortion by vaccinationwith live, attenuated tachyzoites of the NCTS-8 strain of N. caninum.This is the first demonstration of the protection of a pregnant mammalagainst Neospora-induced abortion by vaccination with live, attenuatedtachyzoites derived from a pathogenic strain of Neospora. TABLE 19Geometric mean reciprocal antibody titers (GMT) of pygmy goats 10 dayspost-booster. Reciprocal Antibody Group Titer (GMT) Range A 566  200-6,400 B 283 100-800 C 259 100-400 D <50 <50 E <50 <50

TABLE 20 Protection provided by attenuated live vaccine against fetalabortion induced by N. caninum in pygmy goats. No. Aborted/ PercentGroup Vaccine No. challenged Protection A NC-1 4/4  0 B NCTS-8 2/5 60 CNCTS-8 2/4 50 w/adjuvant D Sham 5/6 17 E pregnancy not challenged n.a.control

Deposit of Biological Materials

The following biological materials were deposited with the American TypeCulture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md. 20852.USA, on Nov. 6, 1996, and were assigned the following accession numbers:

-   -   1. NC-1 strain of Neospora caninum in MARC145 monkey kidney        cells, ATCC Accession No. CRL-12231.    -   2. NCTS-8 strain of N. caninum in MARC145 monkey kidney cells,        ATCC Accession No. CRL-12230.

All patents, patent applications, and publications cited above areincorporated herein by reference in their entirety.

The present invention is not limited in scope by the specificembodiments described, which are intended as single illustrations ofindividual aspects of the Invention. Functionally equivalentcompositions and methods are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the field ofmicrobiology, parasitology, immunology, molecular biology, veterinarymedicine and related fields from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A culture of cells of a strain derived from a pathogenic parentstrain of a species of Neospora, which cells exhibit attenuatedpathogenicity compared to those of the parent strain but which arecapable of triggering an immune response that protects a mammal againstneosporosis when administered as a live vaccine.
 2. (canceled)
 3. Theculture of claim 1, wherein the species of the parent strain is N.caninum.
 4. The culture of claim 3, wherein the parent strain of N.caninum is NC-1 which is present in MARC145 monkey kidney cells havingATCC accession No. CRL12231.
 5. (canceled)
 6. A vaccine to protect amammal against neosporosis, comprising an immunologically effectiveamount of live cells of a strain derived from a pathogenic parent strainof a species of Neospora, which cells exhibit attenuated pathogenicitycompared to those of the parent strain but which are capable oftriggering an immune response that protects the mammal againstneosporosis when administered as a live vaccine, and a veterinarilyacceptable carrier.
 7. (canceled)
 8. The vaccine of claim 6, wherein thespecies of the parent strain is N. caninum.
 9. The vaccine of claim 8,wherein the parent strain of N. caninum is NC-1 which is present inMARC145 monkey kidney cells having ATCC accession No. CRL12231. 10.(canceled)
 11. The vaccine of claim 6, further comprising an adjuvant.12. The vaccine of claim 11, wherein the adjuvant is an oil-in-wateremulsion.
 13. A method for preparing a culture of attenuated cells of aspecies of Neospora for use in a vaccine that protects a mammal againstneosporosis, comprising modifying cells from a pathogenic parent strainof a species of Neospora; selecting and clonally propagating one or moremodified cells that exhibit attenuated pathogenicity compared to cellsof the parent strain; and selecting and clonally propagating one or moreattenuated cells which are capable of triggering an immune response thatprotects the mammal against neosporosis when administered in a livevaccine.
 14. (canceled)
 15. The method of claim 13, wherein the speciesof the parent strain is N. caninum.
 16. The method of claim 15, whereinthe parent strain of N. caninum is NC-1 which is present in MARC145monkey kidney cells having ATCC accession No. CRL-12231.
 17. A methodfor preparing a vaccine to protect a mammal against neosporosis,comprising modifying cells from a pathogenic parent strain of a speciesof Neospora; selecting and clonally propagating those modified cellsthat exhibit attenuated pathogenicity compared to cells of the parentstrain but which are capable of triggering an immune response in themammal that protects against neosporosis when administered in a livevaccine; and combining an immunologically effective amount of theattenuated cells with a veterinarily acceptable carrier in a formsuitable for administration as a live vaccine to the mammal. 18.(canceled)
 19. The method of claim 17, wherein the species of the parentstrain is N. caninum.
 20. The method of claim 19, wherein the parentstrain of N. caninum is NC-1 which is present in MARC 145 monkey kidneycells having ATCC accession No. CRL-12231.
 21. The method of claim 20,wherein the strain of attenuated cells is NCTS-8 which is present inMARC145 monkey kidney cells having ATCC accession No. CRL-12230.
 22. Themethod of claim 17, further comprising adding an adjuvant to thevaccine.
 23. The method of claim 22, wherein the adjuvant is anoil-in-water emulsion.
 24. A method of vaccinating a mammal againstneosporosis, comprising administering to the mammal an immunologicallyeffective amount of a vaccine comprising live cells of a strain derivedfrom a pathogenic parent strain of a species of Neospora, which cellsexhibit attenuated pathogenicity compared to those of the parent strainbut which are capable of triggering an immune response that protects themammal against neosporosis when administered as a live vaccine, and aveterinarily acceptable carrier.
 25. (canceled)
 26. The method of claim24, wherein the species of the parent strain is N. caninum.
 27. Themethod of claim 26, wherein the parent strain of N. caninum is NC-1which is present in MARC145 monkey kidney cells having ATCC accessionNo. CRL-12231.
 28. The method of claim 27, wherein the strain ofattenuated cells is NCTS-8 which is present in MARC145 monkey kidneycells having ATCC accession No. CRL-12230.
 29. The method of claim 24,wherein the vaccine further comprises an adjuvant.
 30. The method ofclaim 29, wherein the adjuvant is an oil-in-water emulsion.
 31. Themethod of claim 24, wherein the mammal is selected from the groupconsisting of dogs, cows, goats, sheep and horses.
 32. A combinationvaccine, comprising an immunologically effective amount of live cells ofa strain derived from a pathogenic parent strain of a species ofNeospora, which cells exhibit attenuated pathogenicity compared to thoseof the parent strain but which are capable of triggering an immuneresponse that protects the mammal against neosporosis when administeredas a live vaccine; one or more antigens that trigger an immune responsethat protects the mammal against a disease or a pathological condition;and a veterinarily acceptable carrier.